The 2,160,837–base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low–guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences inS. pneumoniae that could contribute to differences in virulence and antigenicity.